Chromophore chemistry of fluorescent proteins controlled by light

Abstract

Recent progress in molecular engineering of genetically encoded probes whose spectral properties are controlled with light, such as photoactivatable, photoswitchable and reversibly switchable fluorescent proteins, has brought the new possibilities to bioimaging and super-resolution microscopy. The development of modern photoconvertible proteins is linked to the studies of light-induced chromophore transformations. Here, we summarize the current view on the chromophore chemistry in the photocontrollable fluorescent proteins. We describe both the fundamental principles and the specific molecular mechanisms underlying the irreversible and reversible chromophore photoconversions. We discuss advancements in super-resolution microscopy that became possible due to the engineering of new protein phenotypes and understanding of their chromophore transformations.

Figure 1. Light induced chromophore transformations in irreversibly photoswitchable FPs

Colors highlighting chromophores correspond to the spectral range of observed fluorescence. Non-fluorescent chromophores are shown in gray. [O] denotes an oxidation reaction. (a) Core chromophores of GFP-like proteins, formed by invariant Tyr66, Gly67, and variable residue 65 (the numbering follows that for GFP). (b-h) Chromophore transformations are shown for PAGFP. (b), PAmCherry1 (d), EosFP (g), KikGR (g), and PSmOrange (h) were confirmed by structural data and/or mass-spectrometry. Chromophore transformations shown for PSCFP2 (c), PATagRFP (e), and PAmKate (f) are proposed based on spectral studies. (b-f) The conservative Glu222 residue, which is decarboxylated upon photoactivation, is shown.

Figure 2. Light induced chromophore transformations in reversibly photoswitchable FPs

Colors highlighting chromophores correspond to the spectral range of observed fluorescence. Non-fluorescent chromophores are shown in gray. (a-e) Chromophore transformations shown for Dronpa (a), Padron (b), rsTagRFP (c), and Dreiklang (e) were confirmed by both structural and spectral data. Chromophore transformations for rsCherryRev (c) and rsCherry (d) are proposed based on their spectral properties.

Figure 3. Proposed combinations of photocontrollable FPs for super-resolution microscopy

(a) Green PAGFP and red PATagRFP or PAmCherry1 are suitable for simultaneous two-color PALM. Both FPs are activated by violet light and fluorescence signals of activated FPs are detected in separate channels. Simultaneous imaging is possible because the activated states of two FPs can be dicriminated from the initial states and between each other. (b) Green rsFP Dronpa and red rsTagRFP can be imaged together in a sequential pcSOFI. First, fluctuations of fluorescence from individual rsTagRFP molecules in the off state are detected and analyzed. Then rsTagRFP is bleached to prevent its activation with blue light used for switching off Dronpa. Next, fluctuations of fluorescence from individual Dronpa molecules in the off state are detected and analyzed. (c) Two PSFPs, PSmOrange or PSmOrange2 and PSCFP2, can potentially be imaged sequentially in two-color PALM with no interference of non-activated signal of one FP into the channel of another FP. First, PSmOrange molecules are activated, imaged, localized, and bleached. Orange and far-red fluorescence signals of PSmOrange are distinguishable from PSCFP2 signal. Then PSCFP2 are stochastically activated, imaged and localized. (d) Combinations of currently available green rsFP, such as rsEGFP, with future red rsFP, like rsTagRFP with improved fatigue resistance (denoted as rsTagRFP*), for use insequential two-color RESOLFT. rsTagRFP* is imaged first with rsEGFP in the dark state. Then rsTagRFP* may be bleached to avoid activation of rsTagRFP by blue light. Next, rsEGFP is imaged.